ANALYSIS OF MOLECULAR MECHANISMS FOR NEURONAL POLARITY AND AXON FORMATION BY CRMP-2

CRMP-2分析神经元极性和轴突形成的分子机制

• 批准号: 13680872
• 负责人: INAGAKI Naoyuki
• 金额: $ 2.24万
• 依托单位: NARA INSTITUTE OF SCIENCE AND TECHNOLOGY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680872/
• 关键词: Neuron; Axon; Dendrite; Cell Polarity; CRMP-2; Tublin; Microtubule; Proteome; プロテオーム

Identification of CRMP-2 as a molecule involved in neuronal polarity and axon formationFormation of an axon and dendrites is a fundamental step in neuronal development. In this research, we found that CRMP-2 was enriched in growing axons of cultured hippocampal neurons. Overexpression of CRMP-2 in the cells led to the formation of supernumerary axons without remarkable increase in the total number of neurites. CRMP-2 not only enhanced the formation of multiple axons during the initial stages of axon specification but also induced axon sprouting from already formed dendrites. Furthermore, expression of truncated CRMP-2 mutants suppressed the formation of primary axon in a dominant negative manner. Thus, local concentrations of CRMP-2 in neurites appear to play a critical role in axon induction of hippocampal neurons, thereby establishing and maintaining neuronal polarity.In order to examine the molecular mechanisms for CRMP-2 induced axonal formation, we also investigated the CRMP-2 bin … More ding proteins. Here, we identified two activities of CRMP-2: tublin-heterodimer binding and the promotion of microtuble assembly. CRMP-2 bound tublin dimers with higher affinity than it bound microtubules. Association of CRMP-2 with microtubules was enhanced by tublin polymerization in the presence of CRMP-2. The binding property of CRMP-2 with tubulin was apparently distinct from that of Tau, which preferentially bound microtubules. In neurons, overexpression of CRMP-2 promoted axonal growth and branching. A mutant of CRMP-2, lacking the region responsible for microtuble assembly, inhibited axonal growth and branching in a dominant-negative manner. Taken together, our results suggest that CRMP-2 regulate axonal growth and branching as a partner of the tublin hetereodimer, in a different fashion from traditional MAPs.Search for molecules involved in neuronal polarity and axon formation by protemic strategyWe further started to investigated the molecules involved in axon and polarity formation in addition to CRMP-2 by proteomic strategy. In order to searched molecules involved in axon and polarity formation, we established a 2-DE two-dimensional gel electrophoresis (2-DE) system of highresolution. We utilized fourteen large 2-DE gels (twelve 24 cm x 70 cm gels and two 18 cm x 70 cm gels) which were assembled into a 93 cm x 103 cm cybergel. Our data suggested that the cyber gel can display more than 11,000 protein spots expressed in a 1-10^5 dynamic range in cells. The utilization of present strategy led to about 500% increase in the number of detected spots in comparison to a standard procedure. With this system, we are currently screening molecules which are expressed during the processes of neuronal polarity formation. Less

CRMP-2是参与神经元极性和轴突形成的分子轴突和树突的形成是神经元发育的基本步骤。在本研究中，我们发现CRMP-2在培养的海马神经元的生长轴突中丰富。CRMP-2在细胞中的过表达导致多个轴突的形成，但没有明显增加轴突总数。CRMP-2不仅在轴突指定的初始阶段促进了多个轴突的形成，而且还诱导了已经形成的树突的轴突萌发。此外，截短的CRMP-2突变体的表达以显性负向方式抑制了初级轴突的形成。因此，轴突中CRMP-2的局部浓度似乎在海马神经元轴突诱导中起着关键作用，从而建立和维持神经元的极化。为了研究CRMP-2诱导轴突形成的分子机制，我们还研究了CRMP-2结合…更多的丁蛋白。在这里，我们确定了CRMP-2的两个活性：微管-异二聚体结合和促进微管组装。CRMP-2结合微管蛋白二聚体的亲和力高于其结合的微管。在CRMP-2存在的情况下，微管蛋白聚合增强了CRMP-2与微管的结合。CRMP-2与微管蛋白的结合特性明显不同于Tau，后者优先结合微管。在神经元中，CRMP-2的过表达促进了轴突的生长和分支。CRMP-2的一个突变体，缺乏负责微管组装的区域，以显性-负性方式抑制轴突生长和分支。综上所述，我们的结果表明，CRMP-2作为微管蛋白异源二聚体的伙伴，以不同于传统MAP的方式调节轴突的生长和分支。通过蛋白质组学策略寻找参与神经元极性和轴突形成的分子我们进一步开始利用蛋白质组学策略研究CRMP-2以外参与轴突和极性形成的分子。为了寻找参与轴突和极性形成的分子，我们建立了高分辨率的双向凝胶电泳(2-DE)系统。我们使用了14个大的2-DE凝胶(12个24厘米×70厘米凝胶和两个18厘米×70厘米凝胶)，它们被组装成93厘米×103厘米的网络凝胶。我们的数据表明，这种网络凝胶可以展示11,000多个蛋白质点，在细胞内1-10^5个动态范围内表达。与标准程序相比，本策略的使用导致检测到的斑点数量增加了约500%。利用这个系统，我们目前正在筛选在神经元极性形成过程中表达的分子。较少

项目成果

稲垣直之: "神経ネットワークとシナプスダイナミックス"塩坂貞夫編、金芳堂(in press). (2003)

Naoyuki Inagaki：“神经网络和突触动力学”，由 Sadao Shiosaka、Konpodo 编辑（2003 年出版）。

勝田和大: "Multiple large gel two-dimensional electrophoresis for proteomics"Jap. J. Electrophresis. (In press). (2002)

Kazuhiro Katsuta：“用于蛋白质组学的多重大型凝胶二维电泳”J. Electrophresis（出版中）。

稲垣直之: "細胞内の空間シグナル"細胞工学. 21・4. 345 (2002)

稻垣直之：“细胞内空间信号”细胞工程21・4。

N.Inagaki, et al.: "High resolution large gel two-dimensional electrophoresis for proteomics"BIO forum Int.. 6. 324-325 (2002)

N.Inagaki 等人：“用于蛋白质组学的高分辨率大凝胶二维电泳”BIO 论坛 Int.. 6. 324-325 (2002)

Y.Fukuta, et al.: "CRMP-2 binds to tublin heterodimers to promote microtubule assembly"Nature Cell Biol.. 4. 583-591 (2002)

Y.Fukuta 等：“CRMP-2 结合微管蛋白异二聚体以促进微管组装”Nature Cell Biol.. 4. 583-591 (2002)